Int. J. Vitam. Nutr. Res., 77 (3), 2007, 205Ð216 Effect of Genotype on Micronutrient Absorption and : a Review of Iron, Copper, Iodine and Selenium, and Folates Richard Mithen

Institute of Food Research, Colney Lane, Norwich, NR4 7UA, UK

Received for publication: July 28, 2006

Abstract: For the majority of micronutrients, there are very little data, or none at all, on the role of genetic poly- morphisms on their absorption and metabolism. In many cases, the elucidation of biochemical pathways and regulators of homeostatic mechanisms have come from studies of individuals that have mutations in certain . Other polymorphisms in these genes that result in a less severe phenotype may be important in determining the natural range of variation in absorption and metabolism that is commonly observed. To illustrate some of these aspects, I briefly review the increased understanding of iron metabolism that has arisen from our knowledge of the effects of mutations in several genes, the role of genetic variation in mediating the nutritional effects of io- dine and selenium, and finally, the interaction between a genetic polymorphism in folate metabolism and folic acid fortification.

Key words: Micronutrients, genetic polymorphisms, iron, iodine, selenium, folates

Introduction the interpretation of epidemiological studies, in which some of the variation observed in nutrient status or re- Recently there has been considerable interest in the role quirement may be due to genetic variation at a few or sev- that genetic polymorphisms may play in several aspects eral loci that determine the uptake and metabolism of var- of human nutrition, and the ill-defined terms nutrige- ious nutrients. The increase in interest and knowledge of nomics and nutrigenetics have become commonplace [1]. diet- interactions, largely driven by the expansion of For some researchers, this aspect of nutrition is associat- knowledge on the nature of the , is at a time ed with the suggestion that diets can be personalized Ð that of rapid change in the economic and nutritional status of by knowing one’s genotype it may be possible to fine-tune the population of both developed countries and so-called one’s diet to ensure avoidance of chronic disease. For oth- developing countries. As the health burden of the “west- ers, this science has value in assisting with, for example, ern diet” is rapidly becoming apparent with an increase in

DOI 10.1024/0300-9831.77.3.205 Int. J. Vitam. Nutr. Res., 77 (3), 2007, © Hogrefe & Huber Publishers 206 R. Mithen: Genetic Variation and Micronutrients

such conditions as obesity and type 2 , there is a matosis) [2]. Iron deficiency is of particular significance “westernization” of both the economies and diet of many in early childhood and amongst menstruating women, countries in Asia and south and central America. The eco- whereas hemochromatosis is most prevalent in late-mid- nomic expansion of countries such as China, India, and dle-aged men and post-menopausal women. Hereditary Brazil has led to enormous disparities of both income and hemochromatosis leads to the accumulation of iron and nutritional status within these countries. This is also like- eventually to the deposition of iron in vital organs. The ly to be combined with greater genetic susceptibility of specific genetic defects that cause this disorder have been many people within these countries to conditions such as identified and these provide an insight into the manner by type 2 diabetes. Thus, in many countries that were once which iron uptake is regulated. Other polymorphisms in considered “developing” there are issues concerned with these genes may not result in acute hemochromatosis, but both nutrient deficiency and nutrient excess, at least in may still influence iron uptake. terms of the major macronutrients. In humans, iron is strictly conserved. Each day, about The situation regarding micronutrient status (sensu la- 20 mg of iron is recycled from senescent red blood cells to, to include minerals, vitamins, and phytochemicals) is into the synthesis of new red blood cells (Figure 1A). There probably more complex. The issues of sufficient mi- are no pathways in humans for iron excretion, apart from cronutrient intake to prevent deficiency diseases, such as losses in epithelial cells such as skin, gastrointestinal cells, vitamin D and rickets, have now largely (although not en- and urinary tract cells, and fluids such as blood, tears, and tirely) been displaced in many western countries. This has sweat. Thus, absorption of dietary iron must be regulated been replaced by the realization that the nutrient status for to prevent excess iron accumulation [3]. “optimal” health required for the prevention of chronic Heme iron derived from meat is an important source of disease may be quite different for that to prevent defi- iron and is highly bioavailable, although the precise route ciency. Thus, it is considered by many that the level of mi- of uptake is not understood. It is thought that the metal cronutrients within large sections of the western popula- porphyrin ring is split from the globin in the lumen, and tion is below that required for optimal health. Thus, when the intact Fe porphyrin is transported across the brush bor- considering the importance (or otherwise) of genetic vari- der membrane. There are some suggestions that this may ation in determining micronutrient absorption and metab- happen by endocytosis, and a brush border heme recep- olism, we need to consider both that required to prevent tor, HCP1, has been reported [4]. Within the enterocyte, deficiency diseases, and that required for “optimal” health, ferrous ions may be released from the heme and enter a or the reduction in risk of chronic disease. common pool with non-heme iron [5]. Thus subsequent- For the majority of micronutrients, there are very little ly, the basolateral transfer of iron, and its regulation, may data, or none at all, on the role of genetic polymorphisms be the same as for both heme and non-heme iron (Figure on their absorption and metabolism. In many cases, the 1B). elucidation of biochemical pathways and regulators of The pathway of absorption of non-heme iron is better homeostatic mechanisms have come from studies of indi- understood. Within the lumen, iron is likely to be ferric viduals that have mutations in certain genes. Other poly- ion and is poorly available. Fe3+ ions may be chelated (e.g. morphisms in these genes that result in a less severe phe- with phytate or citrate) or encased as a multi-ion form such notype may be important in determining the natural range as . Fe3+-phytate is poorly absorbed, but ferritin may of variation in absorption and metabolism that is com- be absorbed, although the precise route is yet to be dis- monly observed. To illustrate some of these aspects, I covered. Ferric reductase activity within the duodenal mu- briefly review the increased understanding of iron metab- cosa converts Fe3+ to Fe2+ ion. The cytochrome B DcytB olism that has arisen from our knowledge of the effects of is the most likely candidate for the ferric reductase, but mutations in several genes, the role of genetic variation in the lack of phenotype with DcytB knockout mice suggests mediating the nutritional effects of iodine and selenium, that there may be other ferric reductases [5]. Ferrous ions and finally, the interaction between a genetic polymor- are transported into enterocytes via the divalent metal phism in folate metabolism and folic acid fortification. transporter (DMT1, also known as DCT1 or Nramp2). It seems likely that DMT1 can also transport other divalent ions such as Zn2+, Co2+, Mn2+, and Cd2+, although the phys- iological significance of this is not clear, and these ions Polymorphism in genes affecting may have other transport routes into enterocytes. Once inorganic iron uptake and transport within the enterocyte Fe2+ may either be bound to ferritin or exported into the bloodstream (Figure 1B). The trans- The interaction between diet and genetic factors can lead port of Fe2+ into the circulatory system is due to the coor- to both iron deficiencies and (hemochro- dinated expression and action of the transport protein fer-

Int. J. Vitam. Nutr. Res., 77 (3), 2007, © Hogrefe & Huber Publishers R. Mithen: Genetic Variation and Micronutrients 207

A Senescent red blood cells Spleen Fe, IL-6, O 2 ~ 20 mg Fe /day e- 2+ 3+ 3+ Fe Fe Plasma Fe 2-Tf

20 mg Fe /day Intestinal uptake 1-2 mg Fe /day (Figure 2)

Bone marrow (Figure 3)

Heme Heme receptor (HCP1) ? B Fe

Fe Fe 2+ ?

Fe DcytB HO 1 Fe 3+

2+ 3+ 3+ Ferritin Fe Fe Fe 2-Tf Fe 2+ e- DMT1

Heme Heme receptor (HCP1) ? C Fe liver Fe Fe 2+ ?

Fe DcytB HO Ferroportin 1 Fe 3+ Hepcidin Figure 1: A. Model for the Ferritin Fe 2+ recycling of iron and homeostat- Fe 2+ DMT1 Hephaestin ic control mediate by hepcidin. B. Enterocyte model for the uptake of heme and non-heme iron from the lumen. C. Regula- tion of uptake via hepcidin.

Int. J. Vitam. Nutr. Res., 77 (3), 2007, © Hogrefe & Huber Publishers 208 R. Mithen: Genetic Variation and Micronutrients

roportin 1 (also known as IREG1) and hephaestin on the Mutations in other genes that probably affect hepcidin basolateral surface of the enterocyte [6]. Hephaestin (a Cu- expression can also result in similar phenotypes. For ex- containing protein Ð see below), which has a high degree ample, those in TFR2 are much rarer than the mutations of homology with the serum ferroxidase ceruloplasmin, in HFE, but can result in a similar phenotype. Likewise, results in the oxidation of ferrous to ferric ion, which then the infrequent mutations in HJV, causing acute juvenile binds to (Tf) and is transported in the serum. hemochromatosis. Mutations can also occur in the hep- (Figure 1B). Uptake of Tf-bound iron into cells such as cidin gene (HAMP) itself. the immature erythroid cell for heme synthesis, is via the Mutations in the ferroportin gene can also result in he- Tf receptor (TfR1) through endocytosis. The endosome is mochromatosis [6]. A particular mutation in this gene acidified resulting in release of ferric ion which is reduced (Q248H) occurs within people of African descent at a fre- by Steap3, and possibly other closely related ferric re- quency of about 20% and has been studied both with re- ductases, and then transported out of the endosome by spect to the relatively high frequency (~ 10%) of iron over- DMT1 [7, 8]. The Tf-TfR1 complex is recycled to the cell load amongst adult Africans in sub-Saharan Africa [12], membrane to begin the endocytosis cycle over again. With- and iron deficiency that is common amongst African chil- in macrophages, iron derived from heme is returned to the dren [13]. In neither case was a strong association ob- plasma via ferroportin 1 (as in enterocytes), but is oxidized served. However, there is some evidence that Q248H het- probably via ceruloplasmin as opposed to hephaestin. erozygosity may protect children from iron deficiency The regulation of this entire process is largely depen- when they are exposed to repeated or prolonged inflam- dent upon the protein hepcidin [9]. Hepcidin is a 25-amino matory conditions [13]. acid peptide synthesized in the liver. Hepcidin knockout The hypothesis that mutations in a variety of genes that mice have hemochromatosis, whereas overexpressing result in iron overload were selected for times in which hepcidin in mice leads to iron deficiency. Hepcidin binds diets were low in iron are attractive, but lacking in evi- to ferroportin 1, resulting in it being internalized in the dence. However, it is likely that western diets are richer in cell and degraded (Figure 1C). Thus, this prevents iron be- heme iron than diets in prehistory, and that there is cur- ing transported out of the enterocyte where it accumulates. rently less infection by hookworm and other parasites that The enterocyte only lives for a few days after which it is would have resulted in iron loss through bleeding into the shed from the tips of the villi into the lumen. When iron gastrointestinal tract. Thus, it is conceivable that for most stores are adequate or high, the liver produces hepcidin of prehistory, genes that now result in iron overload may that circulates to the small intestine where it prevents the have been favorably selected. transport of iron from the enterocyte to the plasma. When Hereditary conditions such as hemochromatosis may iron stores are low, hepcidin production is suppressed, fer- provide insights into polymorphisms and mutations that roportin proteins are expressed on the basolateral surface may affect iron homoeostasis. There is a public health con- of the enterocyte and enable iron to be transported into the cern that if food is fortified with iron to prevent deficien- plasma. Hepcidin may have other secondary effects. For cies, then individuals with specific genotypes may absorb example, rising iron content with enterocytes may result inappropriately high amounts of iron. However, interven- in a reduction in expression of DMT1 [9]. tion studies have provided no evidence that C282Y het- The most common form of hereditary hemochromato- erozygotes absorb more dietary iron than those who are sis is due to mutations in the HFE gene [2, 10]. The HFE not carrying the mutation [14, 15]. protein is similar to MHC class I-type proteins and asso- The penetrance of the hemochromatosis phenotype in ciates with beta2-microglobulin (beta2M); the precise role individuals with these mutations is relatively low [16], and of this protein is not understood, but it seems likely it is this has prompted the more systematic survey of seeking involved with the regulation of hepcidin synthesis. The mutations in other genes that affect iron homeostasis. Lee C282Y mutation is most prevalent in northwestern Eu- and colleagues [17] undertook a survey of polymorphisms rope, and it is thought that this mutation arose relatively within 24 genes, all of which have been implicated in recently, maybe between 1000Ð3000 years ago [11]. An homeostasis, and as expected identified many polymor- older mutation, H63D, has a more global distribution. Oth- phisms. As with the more commonly studied polymor- er polymorphisms in this gene have also been described. phisms, these may contribute to variation in iron uptake It seems likely that the C282Y and H63D mutations re- amongst people with no symptoms of iron deficiency or duce the synthesis of hepcidin, which in turn increases the overload. Providing the evidence that this is the case is dif- membrane-located expression of ferroportin leading to ex- ficult, and there is a need to examine several polymor- cess iron uptake. Mutations in HFE are recessive disor- phisms in many genes concurrently. The use of single-nu- ders of low penetrance, and largely affect older men and cleotide polymorphism (SNP) arrays or transcript map- post-menopausal women. ping may both provide useful approaches.

Int. J. Vitam. Nutr. Res., 77 (3), 2007, © Hogrefe & Huber Publishers R. Mithen: Genetic Variation and Micronutrients 209

Copper ly homologous to ATP7B but expressed not in the liver but in the TGN membranes of the placenta, gut, and brain. Copper is required for the activity of several , in- Copper that crosses the TGN membrane in these organs cluding the ferroxidases ceruloplasmin and hephaestin, is required for incorporation into specific enzymes that and cytochrome c oxidase, required for respiration and the may contribute, for example, to the developing brain or to antioxidant Cu, Zn (SOD), which supply a growing fetus. Thus, individuals who suffer from represents about 1% of total cell protein. Its role in fer- Menkes disease are deficient in many essential Cu en- roxidases links it intimately to iron homeostasis [18, 19]. zymes, and die within the first few years of life. Copper is readily absorbed from the diet. Uptake into the Mutations in the ceruloplasmin gene have provided an mucosa cell is probably by passive diffusion. Within the insight into the role of this protein in regulating iron home- enterocyte it may be bound to metallothionein or passes ostasis [22]. Surprisingly, despite 95% of serum Cu being through the basolateral membrane into the bloodstream. in the form of ceruloplasmin, reduced levels of this pro- Within the serum, copper is probably transported bound tein, either due to mutation in the ceruloplasmin gene it- to either albumin or histidine, where it is delivered to tis- self or through another cause (such as Wilson’s disease), sues and the liver for further metabolism. It is transport- does not lead to symptoms of copper deficiency, but in- ed into hepatocytes via the copper transporter, Ctrl. Once stead to anemia. This confirms that ceruloplasmin is not inside the hepatocyte, Cu has four different fates. Firstly, the Cu transport protein in an analogous manner to iron it may be bound to metallothionein, as in the enterocyte and ferritin, and its major role is the mobilization of iron joining an intracellular copper pool. Secondly, it may be from tissues (with the exception of enterocytes) through trafficked into the mitochrondia via the copper chaperone its ferroxidase activity. Several mutations have been doc- cox 17 for incorporation into cytochrome c oxidase. Third, umented in the 20 exons of the ceruloplasmin gene. These it may be bound to CCS (copper chaperone for SOD) for can result in neurological disorders due to iron accumu- Cu, Zn SOD synthesis. Lastly, it may be trafficked to the lation in the basal ganglia, diabetes mellitus resulting from trans Golgi network (TGN) by HAH1 (human atox-1 ho- iron accumulation in the pancreas, and visual degenera- mologue) which delivers it to a P-type ATPase (ATP7B), tion due to iron accumulation in the retina. Ceruloplasmin where it is incorporated into ceruloplasmin, which is trans- does not cross the blood-brain barrier, and thus while the ported into the serum, and is the major form of copper in liver is a major site of ceruloplasmin synthesis, it can al- the serum (> 95%). Alternatively, when Cu concentration so be synthesized in astrocytes. There is evidence from is sufficient or too high, Cu is transported from the TGN Xenopus, that within these tissues, ceruloplasmin may be to a vesicular compartment that migrates towards the bil- synthesized as a glycophosphatidylinositol-linked iso- iary epithelium. This provides a mechanism for removal form, and not excreted. Indeed there is some evidence for of excess Cu in the bile [20]. a rather separate brain iron cycle distinct from that of the Mutations have been described in several of these general circulation. The precise manner by which abnor- genes, and these have been used to further elucidate the malities in ceruloplasmin levels may be associated with genetic and physiological basis of copper metabolism neurological disorders still needs to be resolved [23]. [20]. Mutations in ATP7B cause the autosomal recessive Wilson’s disease, which occurs in about 1:30000 in most populations. Malfunction of ATP7B prevents trafficking of copper within the hepatocyte into the TGN for either Selenium, Iodine and thyroid ceruloplasmin synthesis or excretion via the bile. The con- metabolism sequence is that copper accumulates in the liver, causing oxidative damage, and serum levels of ceruloplasmin are Population effects of severe iodine deficiency, termed io- very low. There may be leakage of Cu from the liver re- dine deficiency disorders (IDDs), include endemic goiter, sulting in Cu deposits in the brain and cornea. The ATP7B hypothyroidism, cretinism, decreased fertility rate, in- gene has 21 exons, and a large number of mutations in this creased infant mortality, and mental retardation. Defi- gene have been described. The most frequent mutation in ciency is an important risk factor for brain damage and the Caucasian population, C3207A, disrupts ATP binding, motor-mental development in the fetus, the neonate, and and occurs in between 40 and 60% of people with Wil- child. 29% of the world’s population is estimated to live son’s disease. This mutation is absent from people of Asi- in areas of deficiency. This occurs primarily in mountain- atic origin [21]. Wilson’s disease can be treated with Cu- ous regions such as the Himalayas, the European Alps, and chelating agents, but can be fatal if not treated. the Andes, where iodine has been washed away by glacia- The related X-linked Menkes disease is caused by a tion and flooding. Iodine deficiency also occurs in low- mutation in ATP7A, which is functionally and structural- land regions far from the oceans, such as central Africa

Int. J. Vitam. Nutr. Res., 77 (3), 2007, © Hogrefe & Huber Publishers 210 R. Mithen: Genetic Variation and Micronutrients

and eastern Europe. It is likely that the extent of iodine also in brown fat. This gene has been shown to have sev- deficiency is more widespread than is usually considered; eral polymorphisms. One of these, A274G, predicts a non- it has been estimated that half of the population of western conservative Thr92Ala substitution has been associated and central Europe suffers from iodine deficiency [24]. with insulin resistance and obesity [27]. Guo and col- The severity of IDD is also dependent upon other factors leagues [28] investigated the potential effect of this poly- Ð such as nutrient status of Vitamin A, iron, and selenium. morphism and two further SNPs, both within non-coding Dietary iodine is taken up readily through the gut in the regions of the DIO2 gene, with mental retardation in an form of iodide. From the circulation, it is concentrated in iodine-deficient area of China. They reported an associa- the thyroid gland by means of an energy-dependent sodi- tion between the occurrences of the two SNPs within non- um-iodate symporter (NIS). In the follicle cells of the thy- coding regions of the DIO2 gene with mental retardation, roid gland, 4 atoms of iodine are incorporated into each and suggested that abnormal DIO2 activity may be of par- molecule of thyroxine (T4) and 3 atoms into each mole- ticular importance in human fetal growth in regions of io- cule of triiodothyronine (T3). T3 is 20Ð100 times more bi- dine deficiency. ologically active than T4. These are essential for neuronal development, sexual development, and growth and for regulating the metabolic rate, body heat, and en- Genetic mutations affecting ergy. T4 is the major product of thyroid secretion and is a incorporation of selenium into critical signal in the plasma that mediates the thyrotropin (TSH) negative feedback loop. When there is insufficient selenoenzymes iodine for thyroid synthesis, the serum T4 level initially falls and the pituitary gland releases TSH, which Severe selenium deficiency occurs in similar areas of the stimulates the growth and metabolic activity of thyroid world to iodine deficiency, such as regions of China, alpine follicular cells. TSH stimulates the thyroid cells to increase Europe, and central Africa. It can cause severe and fatal iodine uptake, and thyroid hormone synthesis and secre- illness, notably Keshan disease, which is a cardiomyopa- tion. However, the stimulating effect of T4 on TSH secre- thy affecting mostly young and middle-aged women. tion is due to the activity of iodothyronine deiodinase Kashin-Beck disease occurs in areas where there is com-

(DIO2), which converts T4 to the biologically active T3. bined iodine and selenium deficiency, such as in areas of This also releases iodine into the circulation, which may Tibet, Siberia, and North Korea. Kashin-Beck disease is either be excreted via the kidneys or reabsorbed by the an osteoarthropathy of the hands and fingers, elbows, thyroid gland. In addition to its role in mediating the neg- knees, and ankles in children and adolescents. It has a com- ative feedback mechanism, DIO2 is of more widespread plex aetiology, and in addition to iodine and selenium, oth- importance in converting T4 to T3 in the brain to enable er abiotic and biotic factors may be involved, and the spe- proper brain development and growth. Thus, polymor- cific role of selenium is yet to be resolved [29Ð31]. Sele- phisms in two critical genes, NIS and DIO2, may be of nium and iodine deficiencies also occur in central Africa, importance for iodine uptake and metabolism. and results in myxedematous cretinism, characterized by NIS is a specialized plasma membrane glycoprotein hypothyroidism from infancy resulting in short stature and that mediates active I- transport into thyroid and other or- mental deficiency [32Ð34]. The cause of this condition is gans, such as the salivary glands, breast, and gastric mu- thought to be due to iodine deficiency, and also dietary cosa. NIS consists of 15 exons and encodes a protein of goitrogens, resulting in increased production of H2O2 in 643 amino acids. About ten NIS mutations have been de- the thyroid due to stimulation by thyrotropin. Inadequate scribed to date [25], Congenital iodine transport disorder glutathione peroxidase activity due to selenium deficien- (ITD) due to one or more of these mutations is an infre- cy prevents removal of excess H2O2, leading to tissue quent autosomal recessive disorder. As with the majority necrosis and resulting in destruction of the gland soon af- of polymorphisms in genes determining uptake and me- ter birth [35]. tabolism of micronutrients, the importance of these mu- In contrast to these examples of selenium deficiencies, tations are only recognized when in a homozygous state there is concern within some western European countries in which they result in irreversible deleterious effects on that the level of selenium in the diet may not be adequate development and acute symptoms of hypothyroidism [26]. for “optimal” health, and a relatively low level of seleni- To what extent the transport of I- may be affected when um has been associated with increased risk of and these mutations are in a heterozygous state and any con- lower immune function [36]. sequences for health have not been systematically inves- Selenium is incorporated into about 20Ð30 proteins, tigated. DIO2 is a selenoenzyme and is particularly im- many of which have antioxidant properties and regulate portant in the brain and pituitary, as discussed above, and cell redox status, such as thioredoxin reductase and glu-

Int. J. Vitam. Nutr. Res., 77 (3), 2007, © Hogrefe & Huber Publishers R. Mithen: Genetic Variation and Micronutrients 211

A

२ Sec SPB2 EFSec

AUG AUGUAA U UAA

Sec Sec

Sec Sec Sec

B Mutant २ Sec SPB2 EFSec

AUG AUGUAA U UAA

Sec Sec Figure 2: A. Model for the Sec incorporation of Se into selenoproteins via the SBP2. ‘Essential’ selenoproteins ‘Non-essential’ selenoproteins B. Mutations in SPB2 results in e.g. thioredoxin reductase, plasma e.g. DIO2, selenoprotein P differential incorporation of Se into “essential” and “non- glutathione peroxidase essential” proteins (from reference [40]). tathione peroxidases, while others are involved in trans- sion of the different selenoproteins. These differences are port of selenium to peripheral tissues through plasma se- due in part to the differential ability of tissues and organs lenoprotein P. As discussed above, selenium is also in- to retain Se under conditions of dietary limitations. For corporated into iodothyronine deiodinases, such as DIO2. example, brain and endocrine organs preserve Se better Mutations in rodents that abolish global selenoprotein syn- than liver, kidney, muscles, and blood. However, even thesis are lethal, whereas knockout of specific selenopro- within tissues that are able to retain Se, some selenopro- teins have varying effects. For example, knockout of teins decrease at faster rates than others. thioredoxin reductase is lethal in embryos [37], knockout Selenium is incorporated into proteins through an un- of cytosolic glutathione peroxidase increases sensitivity usual mechanism in which the UGA stop codon is re-cod- to , and knockout of DIO2 results in ab- ed to be read as a sense codon (Figure 2). This is through normal neurological development [38]. Deficiencies in di- part of the 3’ untranslated part of the mRNA of seleno- etary selenium do not cause equal reductions in expres- proteins, termed selenocysteine insertion sequences

Int. J. Vitam. Nutr. Res., 77 (3), 2007, © Hogrefe & Huber Publishers 212 R. Mithen: Genetic Variation and Micronutrients

(SECIS) elements, recruiting a SECIS binding protein nective-tissue abnormalities. Folate-deficient diets pre- (SBP2), which serves as a scaffold for recruiting a se- conception and during pregnancy can lead to neural tube lenocysteine specific elongation factor, EFSec and defects. This has led to both provision of advice in many tRNAsec. Once assembled, this complex mediates the in- countries to either consume a folate-rich diet or to take corporation of selenocysteine at UGA codons [39, 40]. folic acid supplements, which has had no impact on the Polymorphisms in the 3’ untranslated region near to the incidence of NTD, or has led to the mandatory fortifica- SECIS element may be expected to alter the efficiency of tion of flour with folic acid, in a similar manner to niacin, Se incorporation into the specific selenoprotein. For ex- which has resulted in a reduction in the incidence of neur- ample, there is an SNP at position 718 in the GPX4 gene, al tube defects. which is near the SECIS element. In the UK population, Functional polymorphisms have been described in sev- 26Ð33% have a CC genotype whereas 22Ð24% have a TT eral genes concerned with folic acid and folate metabo- genotype at this position. The former have a higher level lism. The most well studied one is the C677T polymor- of lymphocyte 5’-lipoxygenase products, which would be phism in the 5, 10-methylenetetrahydrofolate reductase consistent with the SNP being functionally active, al- (MTHFR) gene. The MTHFR catalyzes the con- though the effect of this polymorphism on GPX4 activity version of 5,10-methylenetetrahydrofolate (methylene- requires further study [41, 42]. Rare mutations in SBP2 THF) to 5-methylenetetrahydrofolate, which is the carbon have been shown to have differential effects on the incor- donor for the methylation of homocysteine to methionine. poration of Se into different selenoproteins [40]. The most The C677T alteration converts an alanine to a valine and severely affected protein was DIO2, with lesser effects on is associated with about a 70% reduction in activity and GPX and selenoprotein P. This may be because the UGA increased thermolability of the enzyme, and an increase codon of DIO2 is most distant from the SECIS element in homocysteine, as the conversion to methionine is less and the half-life of the protein is less than 45 minutes. efficient (Figures 3 and 4). As a consequence, when di- etary folate levels are low, homozygosity for MTHFR 677TT is associated with enhanced risk of neural tube defects [43], vascular disease [44, 45], late-onset Nutrient status, food fortification Alzheimer’s [46], migraine [47], cervical intraepithelial and genetic polymorphism: B vita- neoplasia [48], esophageal [49], and endometrial [50] can- cer. However, the same genotype is also associated with mins, folic acid and the MTHFR reduced risk of colon cancer [51] and acute lymphatic C667T polymorphism leukemia [52], probably due to the contribution of en- hanced supply of methylene-THF to enable proper DNA

The B group of vitamins, including folate, vitamin B12, synthesis and to avoid the misincorporation of dUMP in- niacin (B3), pyridoxal phosphate (B6), and riboflavin (B2) to DNA. have historically been of major interests in the context of The frequency of the MTHFR 677T allele varies sub- overt deficiency diseases such as pellagra and anemia. One stantially in different regions of the world and amongst of the first food fortification programs was the addition of different ethnic groups. It is low amongst sub-Saharan niacin to flour to eradicate pellagra in the United States in Africans (0.07) and Canadian Inuit (0.06) but higher the 1930s. More recently, the importance of the B vita- amongst Caucasians, Japanese, and Chinese (0.24Ð0.54). mins in general and folate in particular in maintaining There is evidence of a north-south increase in allele fre- health and avoiding a wide range of chronic illness Ð in- quency in Europe, and a north-south decrease in frequen- cluding birth defects, cancer, cardiovascular disease, and cy in China [53]. The variation in the frequency of the dementia Ð has been recognized, and that apparently mar- 677T allele raises the question of whether this is a recur- ginal deficiencies in the diet may have health conse- rent mutation or if the allele has a common ancestral ori- quences. The complex effects that folate and other B vit- gin. Through the quantification of frequencies of nu- amins have on health is due to their effects on several cleotide polymorphisms in introns, it seems likely that the processes that contribute to genomic stability and DNA MTHFR 677T allele has a common origin [54], and that repair, including the supply of nucleotides for DNA syn- the variation in frequency may be due to selection, with thesis and methyl groups for epigenetic regulation, com- there being less selection pressure against the 677TT geno- bined with the role of folate and B12 in the conversion of type in regions with a higher folate content to the diet. homocysteine to methionine (Figure 3). Homocysteine is Food fortification with folic acid in the United States a risk factor for vascular pathology Ð such as promotion has reduced the incidence of neural tube defects (NTD) of clot formation and blocked blood vessels, and inhibi- [55], and has also resulted in modest reduction in other tion of collagen-elastin cross-linking that can lead to con- birth defects, such as cleft palate, transposition of the great

Int. J. Vitam. Nutr. Res., 77 (3), 2007, © Hogrefe & Huber Publishers R. Mithen: Genetic Variation and Micronutrients 213

CpG island methylation of DNA

X-CH X Other methylation reactions 3

S-Adenosylhomocysteine S-Adenosylmethionine

Methylation Thromboembolic and Homocysteine cycle Methionine atherogenic vascular disease

MS and MSR

Methylcob(II) alamin Cob(I)alamin Vitamin

B12 cycle

DHFR H folate 4 Cob(II) alamin

Purine biosynthesis → RNA, DNA S H M T 5-methyl-H4folate Natural diet

MTHFR

Methenyl-formylH4folates 5,10-Methylene-H4folate Uracil misincorporation dUMP and DNA instability dTMP H2folate DNA synthesis DHFR

Folic acid Supplement/fortification Figure 3: Folate metabolism.

arteries, and upper limb defects [56]. In contrast, in gen- Methionine eral, dietary recommendations to take folic acid supple- Methionine ments or to have a high folate diet does not appear to have synthase Homocysteine been successful in reducing birth defects in many coun- 5-methyl-H folate tries, leading to calls for increased mandatory fortifica- 4 tion, particularly within developing countries [57]. Fetuses that are either 677TT or 677TC are at higher 667T allele 667C allele risk of NTD [58,59], and are more likely to abort. How- MTHFR ever, analyses of frequency of NTD and MTHFR geno- type between 1988 and 1994, and between 1994 and 1998 show striking changes in risk. During the earlier time pe- riod, there was a far greater risk of a 677TC heterozygote 5,10-Methylene-H4folate suffering NTD, compared to the 677CC homozygotes Thymidylate dUMP (risk ratio 9.8, 2.8Ð34.0 95%CI), but after 1994 there was synthase dTMP no significant difference in risk between these two geno- types [60]. The sample sizes for the 677TT homozygotes were too small to obtain statistical significance, but they Nucleotide and exhibited a similar trend. The explanation may be that be- DNA biosynthesis ginning around 1994 there was strong encouragement by physicians for women to take folic acid supplements pre- Figure 4: The effect of the MTHFR C667T polymorphisms on and post- conception, and this additional folate acid re- the relative flux of folate into DNA synthesis and as a donor of verses the abnormal folate metabolism caused by the 677T methyl groups for methionine biosynthesis. variant. In a similar manner, Muñoz-Morgan and col-

Int. J. Vitam. Nutr. Res., 77 (3), 2007, © Hogrefe & Huber Publishers 214 R. Mithen: Genetic Variation and Micronutrients

leagues [61] reported an increase in the frequency of the 8. Ohgami, R.S., Campagna, D.R., Greer, E.L., Antiochos, B., 677T allele in people under 20 years of age in Spain, and McDonald, A., Chen, J., Sharp, J.J., Fujiwara, Y., Barker, J.E. an increase in the frequency of the 677TT homozygous and Fleming, M.D. (2005) Identification of a ferrireductase genotype from 13% in people > 20 years to 26% in peo- required for efficient transferrin-dependent iron uptake in ple less than 20 years. This data is consistent with an in- erythroid cells. Nat. Genet. 37, 1264Ð1269. 9. Ganz, T. and Nemeth, E. (2006) Iron imports. IV. Hepcidin crease in pregnant women taking folic acid supplements and regulation of body iron metabolism. Am. J. Physiol. Gas- in the early 1980s Ð going from 10% in 1977 to 55% in trointest. Liver Physiol. 290, G199Ð203. 1986. 10. Beutler, E. (2003) Penetrance of haemochromatosis. Gut 52, Thus, it seems probable that dietary advice when adopt- 610Ð611. ed and mandatory folic acid fortification of food is lead- 11. Lucotte, G. and Dieterlen, F. (2003) A European allele map ing to a rapid increase in the frequency of the MTHFR of the C282Y mutation of hemochromatosis: Celtic versus 677T allele. What might the consequences of this be? Viking origin of the mutation? Blood Cells Mol. Dis. 31, There is evidence that the 677T allele increases the ho- 262Ð267. mocysteine level in the blood, and this has been associat- 12. McNamara, L., Gordeuk, V.R. and MacPhail, A.P. (2005) ed with a wide range of degenerative and chronic diseases, Ferroportin (Q248H) mutations in African families with di- including cardiovascular disease and Alzheimer’s disease, etary iron overload. J. Gastroenterol. Hepatol. 20, 1855Ð1858. particularly if folate levels are less than optimal in the di- 13. Kasvosve, I., Gomo, Z.A., Nathoo, K.J., Matibe, P., Mu- et. 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